Dye labeled imidazoquinoline compounds

ABSTRACT

Dye labeled imidazonaphthyridine, imidazopyridine and imidazoquinoline compounds having immune response modulating activity are disclosed. The compounds are useful, inter alia, for determining the binding and/or receptor sites of the molecules.

FIELD OF THE INVENTION

[0001] The invention relates to imidazonaphthyridine, imidazopyridineand imidazoquinoline compounds that have immune response modulatingactivity and that contain a dye moiety, in particular, a fluorescent dyemoiety. The invention also relates to methods of preparing the dyelabeled compounds.

BACKGROUND OF THE INVENTION

[0002] Compounds that are labeled or tagged have long been used in thechemical and biological sciences. Such compounds can be used in avariety of ways. For example, by labeling a compound that is known to bebiologically active, one can more readily identify metabolites of thecompound, one can determine the binding and/or receptor sites for themolecule, one can determine how long the compound remains in the body orother system, and so on.

[0003] One known way to label compounds is by attaching a dye marker tothe compound. This is typically done by grafting a dye moiety onto thebiologically active molecule or by incorporating the dye moiety into thebiologically active molecule during its synthesis. It is important thatthe labeled compound retain the critical properties of the unlabeledcompound such as selective binding to a receptor or nucleic acid,activation or inhibition of a particular enzyme, or ability toincorporate into a biological membrane. There are a wide variety of dyemoieties available, including for example, dipyrrometheneborondifluoride dyes, fluorescein, fluorescein derivatives, rhodamine,rhodamine derivatives and Texas Red.

[0004] The imidazonaphthyridines, imidazopyridines and imidazoquinolinesare part of a unique class of immune response modifier compounds thathave the ability to induce the biosynthesis of interferon and othercytokines. See, for example, Gerster, U.S. Pat. No. 4,689,338; Gersteret al., U.S. Pat. No. 4,929,624; Gerster, U.S. Pat. No. 5,268,376;Gerster et al., U.S. Pat. No. 5,389,640; Nikolaides et al., U.S. Pat.No. 5,352,784; Lindstrom et al., U.S. Pat. No. 5,494,916.; andInternational Publication WO 99/29693. Dyes, particularly fluorescentdyes, are typically relatively large, bulky molecules and it is possiblethat such a large substituent may impair the compound's ability to bindor otherwise interact with the subject cells in a manner that causesbiologic response.

SUMMARY OF THE INVENTION

[0005] We have discovered a class of dye labeled imidazonaphthyridine,imidazopyridine or imidazoquinoline compounds that retain their abilityto induce cytokines. These compounds employ a spacer group to separatethe dye moiety from the active core of the compound so that the bulkydye group does not interfere with the biological activity of themolecule. The compounds of the invention have the generic formula (I):

[0006] wherein:

[0007] R₁ is a spacer group;

[0008] R₂ is hydrogen, alkyl, hydroxyalkyl, haloalkyl, aminoalkyl,alkylaminoalkyl, dialkylaminoalkyl, amidoalkyl, alkylamidoalkyl,dialkylamidoalkyl, alkanoylalkyl, azidoalkyl, carbamoylalkyl, alkyloptionally interrupted by a heteroatom; alkenyl, alkenyloxyalkyl;cycloalkylalkyl, heterocycloalkyl; aryl, aralkyl, aralkenyl,heteroarylalkyl, in which aryl is optionally substituted by alkyl of 1to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, halo, amino,alkylamino or dialkylamino; aroylalkyl, or heteroaroylalkyl;

[0009] R₃ and R₄ are each independently hydrogen, alkyl, alkoxy of 1 to4 carbon atoms, halo, amino, alkylamino, dialkylamino, or when takentogether, R₃ and R₄ form a fused aryl or heteroaryl group that isoptionally substituted by one or more substituents selected from alkylof 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, halo, amino,alkylamino, dialkylamino, hydroxy and alkoxymethyl; or

[0010] R₃ and R₄ form a fused 5- to 7-membered saturated ring,optionally containing one or more heteroatoms and optionally substitutedby one or more substituents selected from alkyl of 1 to 4 carbon atoms,amino, halo and haloalkyl of 1 to 4 carbon atoms; and

[0011] DYE is a dye moiety, with the proviso that the dye moiety is notdansyl; or a pharmaceutically acceptable acid addition salt thereof.

[0012] The invention additionally provides methods of preparing the dyelabeled imidazonaphthyridine, imidazopyridine and imidazoquinolinecompounds.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a histogram plot of the fluorescence intensity fromcells incubated with fluorescent dye alone.

[0014]FIG. 2 is a histogram plot of the fluorescence intensity fromcells incubated with a labeled compound of the invention where the labelis the dye used in the incubation of FIG. 1.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0015] As mentioned above, the invention provides dye labeled immuneresponse modifying compounds of formula (I):

[0016] wherein:

[0017] R₁is a spacer group;

[0018] R₂ is hydrogen, alkyl, hydroxyalkyl, haloalkyl, aminoalkyl,alkylaminoalkyl, dialkylaminoalkyl, amidoalkyl, alkylamidoalkyl,dialkylamidoalkyl, alkanoylalkyl, azidoalkyl, carbamoylalkyl, alkyloptionally interrupted by a heteroatom; alkenyl, alkenyloxyalkyl,cycloalkylalkyl, heterocycloalkyl; aryl, aralkyl, aralkenyl,heteroarylalkyl, in which aryl is optionally substituted by alkyl of 1to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, halo, amino,alkylamino or dialkylamino; aroylalkyl, or heteroaroylalkyl;

[0019] R₃ and R₄ are each independently hydrogen, alkyl, alkoxy of 1 to4 carbon atoms, halo, amino, alkylamino, dialkylamino, or when takentogether, R₃ and R₄ form a fused aryl or heteroaryl group that isoptionally substituted by one or more substituents selected from byalkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, halo,amino, alkylamino, dialkylamino, hydroxy and alkoxymethyl; or

[0020] R₃ and R₄ form a fused 5- to 7-membered saturated ring,optionally containing one or more heteroatoms and optionally substitutedby one or more substituents selected from alkyl of 1 to 4 carbon atoms,amino, halo and haloalkyl of 1 to 4 carbon atoms; and

[0021] DYE is dye moiety, with the proviso that the dye moiety is notdansyl; or a pharmaceutically acceptable acid addition salt thereof.

[0022] In this document, the following terms have the meanings assignedto them below unless otherwise noted:

[0023] Alkyl and alkenyl groups contain from 1 to 8 (or 2 to 8) carbonatoms and may be straight chain or branched. Cycloalkyl groups cancontain from 3 to 8 ring members and may be optionally substituted byalkyl groups. Heterocyclic groups can contain from 3 to 8 ring membersand from 1 to 3 heteroatoms independently selected from O, S, and N.

[0024] Aryl groups are carbocyclic aromatic rings or ring systems.Heteroaryl groups are aromatic rings or ring systems that contain from 1to 6 heteroatoms independently selected from O, S, and N. A preferredaryl group is benzene. Preferred heteroaryl groups are single rings thathave 5 or 6 members and 1 to 4 heteroatoms independently selected fromO, S and N.

[0025] Heteroatoms are O, S, or N.

[0026] The term “oyl” is used to indicate the presence of a carbonylgroup in the radical. For example, “aroyl” is used to refer to anaromatic group that is attached by a carbonyl group to the remainder ofthe structure.

[0027] The spacer group is an organic linking group that allows a dyemoiety to be attached to an imidazonaphthyridine, imidazopyridine orimidazoquinoline compound without substantially reducing its biologicalactivity. Although the invention is not bound by any theory ofoperation, it is thought that the spacer group places enough distancebetween the active core of the molecule and the bulky dye moiety suchthat the dye moiety does not interfere with the interactions between theactive core and the cells that result in cytokine induction. The spacergroup can therefore be any divalent organic linking group that does notitself interfere with the biological activity of the molecule and thatallows a dye moiety to be included in the molecule without substantiallyreducing its biological activity. In this context, a compound'sbiological activity has not been significantly impaired if the labeledcompound induces interferon or tumor necrosis factor biosynthesis whentested at a concentration less than or equal to about 50 μg/ml accordingto Test Method 1 provided below.

[0028] One preferred spacer group has the structural formula (II):

[0029] Preferably, when the spacer group has formula (II) the methylenegroup that is outside the brackets is attached to the dye moiety.

[0030] Another preferred spacer group has the structural formula (III):

[0031] The dye moiety can be derived from any of the known dyes,particularly fluorescent dyes, with the proviso that the dye moiety isnot dansyl. Examples of suitable types of dyes includedipyrrometheneboron difluoride dyes, fluorescein, fluoresceinderivatives, rhodamine, rhodamine derivatives and Texas Red. Manydipyrrometheneboron difluoride (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) dyes are known, see for example, Haugland,et al, U.S. Pat. No. 4,774,339; Kang, et al. U.S. Pat. No. 5,187,288;Haugland et al., U.S. Pat. No. 5,248,782; and Kang et al., U.S. Pat. No.5,274,113. Many of the dipyrromethenboron difluoride dyes arecommercially available from Molecular Probes, Inc., Eugene, Oreg. underthe tradename BODIPY® fluorophores. Preferred dye moieties includefluorescein and 4,4-difluoro-5,7dimethyl-4-bora -3a,4a-diaza-s-indacenewhich has the following structure.

[0032] Preferred compounds of formula (I) includeN-[2-(4-amino-2-ethoxymethyl-1H-imidazo[4,5-c]quinolin-1l-yl)ethyl]-6-[(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino]hexanoamidewhich has the following structure:

[0033] 5-{[({4-[4-amino-2-(2-methoxethyl)-1H-imidazo[4,5-c]quinolin-1-yl]lbutyl}amino)carbonthioyl]amino}-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acidwhich has the following structure:

[0034] and5-{[({2-[4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl]ethyl}amino)carbonthioyl]amino}-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acidwhich has the following structure:

[0035] Compounds of the invention may be prepared according to themethod shown in Reaction Scheme I below. An imidazonaphthyridine,imidazopyridine or imidazoquinoline of Formula IV is reacted with a dyederivative of Formula V to provide a compound of Formula I. R_(A) andR_(B) both contain functional groups which are selected to react witheach other. For example, if R_(A) contains a primary amine, then a dyederivative wherein R_(B) contains an acyl azide, aldehyde, anhydride,carbonyl halide, halide, haloacetamide, imido ester, isocyanate,isothiocyanate; maleimide, succinimidyl ester or sulfonyl chloride isselected. R_(A) and R_(B) are selected such that they react to providethe desired spacer group R₁ (e.g., If R_(A) is —CH₂CH₂NH₂ and R_(B) is—(CH₂)₂C(O)NH(CH₂)₅COOH then R₁ will be—CH₂CH₂NHC(O)(CH₂)₅NHC(O)(CH₂)₂—). Methods for preparing compounds ofFormula IV where R_(A) contains a functional group are known. See forexample, Gerster, U.S. Pat. No. 4,689,338; Gerster et al., U.S. Pat. No.4,929,624; Gerster, U.S. Pat. No. 5,268,376; Gerster et al., U.S. Pat.No. 5,389,640; Nikolaides et al., U.S. Pat. No. 5,352,784; Lindstrom etal., U.S. Pat. No. 5,494,916; Andre, et. al, U.S. Pat. No. 4,988,815;Gerster, U.S. Pat. No. 5,367,076; Gerster, U.S. Pat. No. 5,175,296;Nikolaides et. al., U.S. Pat. No. 5,395,937; Gerster et. al., U.S. Pat.No. 5,741,908; Lindstrom, U.S. Pat. No. 5,693,81 1; Nanba et al., U.S.Pat. No. 6,069,149, the disclosures of which are incorporated byreference herein. See also, International Publication WO 99/29693. Manydye derivatives containing a reactive functional group are commerciallyavailable (e.g. BODIPY® fluorophores, fluorescein isothiocyanate,5-carboxyfluorescein) or may be prepared by known synthetic routes. Seefor example, Haugland, et al, U.S. Pat. No. 4,774,339; Kang, et al. U.S.Pat. No. 5,187,288; Haugland et al., U.S. Pat. No. 5,248,782;and Kang etal., U.S. Pat. No. 5,274,113, the disclosures of which are incorporatedby reference herein. The reaction will generally be conducted bycombining a solution of the compound of Formula IV in a suitable solventsuch as pyridine or dimethyl sulfoxide with a solution of the dyederivative of Formula V in a suitable solvent such as pyridine ordimethylsulfoxide. The reaction may be run at ambient temperature or atan elevated temperature. The product of Formula I is then isolated andpurified using conventional methods.

[0036] The examples below are provided to illustrate the invention, butare not intended to limit it in any way.

CYTOKINE INDUCTION IN HUMAN CELLS—TEST METHOD 1

[0037] An in vitro human blood cell system is used to assess cytokineinduction by compounds of the invention. Activity is based on themeasurement of interferon and tumor necrosis factor (α) (IFN and TNF,respectively) secreted into culture media as described by Testerman et.al. in “Cytokine Induction by the Immunomodulators Imiquimod andS27609”, Journal of Leukocyte Biology, 58, 365-372 (Sep., 1995).

[0038] Blood Cell Preparation for Culture

[0039] Whole blood from healthy human donors is collected byvenipuncture into EDTA vacutainer tubes. Peripheral blood mononuclearcells (PBMCs) are separated from whole blood by Histopaque®-1077 (SigmaChemicals, St. Louis, Mo.) density gradient centrifugation. The PBMCsare washed twice with Hank's Balanced Salts Solution (Sigma) and arethen suspended at 2×10⁶ cells/mL in RPMI 1640 medium containing 10%fetal bovine serum, 2 mM L-glutamine and 1% penicillin/streptomycinsolution (RPMI complete). 1 mL portions of PBMC suspension are added to12 or 24 well flat bottom sterile tissue culture plates.

[0040] Compound Preparation

[0041] The compounds are solubilized in ethanol, dimethyl sulfoxide orpyrogen free water then diluted with tissue culture water, 0.01N sodiumhydroxide or 0.01N hydrochloric acid (The choice of solvent will dependon the chemical characteristics of the compound being tested.). Ethanolor DMSO concentration should not exceed a final concentration of 1% foraddition to the culture wells. The compounds are generally tested usinga concentration range from about 0.01 μg/mL to about 50 μg/mL.

[0042] Incubation

[0043] The solution of test compound is added to the wells containing 1ml of PBMCs in media. The plates are covered with plastic lids, mixedgently and then incubated for 24 hours at 37° C. with a 5% carbondioxide atmosphere.

[0044] Separation

[0045] Following incubation the cell-free culture supernatant is removedwith a sterile polypropylene pipet and transferred to a 12×75 mmpolypropylene tube. The tubes are then centrifuged at 1000 rpm (˜800 xg)for 10 to 15 minutes at 4° C. The supernatant is removed and placed into2 mL sterile freezing vials. Samples are maintained at −70° C. untilanalyzed for cytokines.

[0046] Interferon Analysis/Calculation

[0047] Interferon concentrations are determined by bioassay using A549human lung carcinoma cells challenged with encephalomyocarditis. Thedetails of the bioassay method have been described by G. L. Brennan andL. H. Kronenberg in “Automated Bioassay of Interferons in Micro-testPlates”, Biotechniques, June/July, 78, 1983, incorporated herein byreference. Briefly stated the method is as follows: A549 cells areincubated with dilutions of IFN standard or test samples at 37° C. for24 hours. The incubated cells are then infected with an inoculum of *encephalomyocarditis virus. The infected cells are incubated for anadditional 24 hours at 37° C. before quantifying for viral cytopathiceffect. The viral cytopathic effect is quantified by staining of thewells with a vital dye such as crystal violet followed by visual scoringof the plates. Results are expressed as alpha reference units/mL basedon the value obtained for an NIH Human Leukocyte IFN standard.

[0048] Tumor Necrosis Factor (α) Analysis

[0049] Tumor necrosis factor (α) (TNF) concentration is determined usingan ELISA kit available from Genzyme, Cambridge, Mass. The results areexpressed as pg/ml.

CYTOKINE INDUCTION IN HUMAN CELLS—TEST METHOD 2

[0050] An in vitro human blood cell system is used to assess cytokineinduction. Activity is based on the measurement of interferon and tumornecrosis factor (α) (IFN and TNF, respectively) secreted into culturemedia as described by Testerman et. al. In “Cytokine Induction by theImmunomodulators Imiquimod and S-27609”, Journal of Leukocyte Biology,58, 365-372 (September, 1995).

[0051] Blood Cell Preparation for Culture

[0052] Whole blood from healthy human donors is collected byvenipuncture into EDTA vacutainer tubes. Peripheral blood mononuclearcells (PBMCs) are separated from whole blood by density gradientcentrifugation using Histopaque®-1077. The PBMCs are washed twice withHank's Balanced Salts Solution and then are suspended at 3-4×10⁶cells/mL in RPMI complete. The PBMC suspension is added to 48 well flatbottom sterile tissue culture plates (Costar, Cambridge, Mass. or BectonDickinson Labware, Lincoln Park, N.J.) containing an equal volume ofRPMI complete media containing test compound.

[0053] Compound Preparation

[0054] The compounds are solubilized in dimethyl sulfoxide (DMSO). TheDMSO concentration should not exceed a final concentration of 1% foraddition to the culture wells. The compounds are generally tested atconcentrations ranging from 0.12 to 30 μM.

[0055] Incubation

[0056] The solution of test compound is added at 60 μM to the first wellcontaining RPMI complete and serial 3 fold dilutions are made in thewells. The PBMC suspension is then added to the wells in an equalvolume, bringing the test compound concentrations to the desired range(0.12 to 30 μM). The final concentration of PBMC suspension is 1.5-2×10⁶cells/mL. The plates are covered with sterile plastic lids, mixed gentlyand then incubated for 18 to 24 hours at 37° C. in a 5% carbon dioxideatmosphere.

[0057] Separation

[0058] Following incubation the plates are centrifuged for 5-10 minutesat 1000 rpm (˜200 ×g) at 4° C. The cell-free culture supernatant isremoved with a sterile polypropylene pipet and transferred to sterilepolypropylene tubes. Samples are maintained at −30 to −70° C. untilanalysis. The samples are analyzed for interferon (α) and for tumornecrosis factor (α) by ELISA

[0059] Interferon (α) and Tumor Necrosis Factor (α) Analysis by ELISA

[0060] Interferon (α) concentration is determined by ELISA using a HumanMulti-Species kit from PBL Biomedical Laboratories, New Brunswick, N.J.Results are expressed in pg/mL.

[0061] Tumor necrosis factor (α) (TNF)concentration is determined usingELISA kits available. from Genzyme, Cambridge, Mass.; R&D Systems,Minneapolis, Minn.; or Pharmingen, San Diego, Calif. Results areexpressed in pg/mL.

Preparation of an Unlabeled Compound of Formula IV

[0062]2-(4-Amino-2-ethoxymethyl-1H-imidazo[4,5-c]quinolin-1-yl)ethaneaminetrihydrochloride

[0063] Part A

[0064] Thionyl chloride (32.3 mL, 0.4338 mole) and N,N-dimethylformamide(32 mL, 0.4338 mole) were added sequentially to a suspension of4-hydroxy-3-nitroquinoline (75 g, 0.3944 mole) in dichloromethane (750ml). The reaction mixture was heated at reflux for about 2½ hours andthen held at ambient temperature overnight. The reaction mixture waschilled in an ice bath and then a mixture of triethylamine (82.5 mL,0.5916 moles) and ethanolamine (35.7 mL, 0.5916 mole) in dichloromethanewas slowly added. The reaction mixture was heated at reflux for severalhours and then an additional 0.5 equivalents of both triethylamine andethanolamine were added. The reaction mixture was refluxed for anadditional hour then held at ambient temperature overnight. Theresulting solid was isolated by filtration, washed first withdichloromethane then with water, and dried to provide 75 g of2-[(3-nitro-4-quinolinyl)amino]ethanol.

[0065] Part B

[0066] 2-[(3-Nitro4-quinolinyl)amino]ethanol (6 g) was combined withethanol (200 mL) and platinum on carbon catalyst. The mixture washydrogenated on a Parr apparatus. This procedure was repeated fouradditional times using a total of 30 g of starting material. Themixtures from all five hydrogenations were combined and then filteredthrough a layer of Celite® filter aid to remove the catalyst. Thefiltrate was concentrated under vacuum to provide crude2-[(3-amino-4-quinolinyl)amino]ethanol.

[0067] Part C

[0068] The crude material from Part B was combined with ethoxyaceticacid (13.4 g). The mixture was heated using an oil bath until thereaction was complete. The mixture was cooled to ambient temperature andthen diluted with water and made basic with sodium hydroxide (6N). Themixture was extracted 3 times with dichloromethane. The extracts werecombined, washed with water, dried with magnesium sulfate and thenfiltered. The filtrate was concentrated under vacuum to provide 30.8 gof crude 2-ethoxymethyl-1H-imidazo[4,5-c]quinoline-1-ethanol.

[0069] Part D

[0070] Peracetic acid (25 mL of 32%) was added to a mixture of thematerial from Part C and methyl acetate (350 mL). The reaction mixturewas heated at 54° C. until thin layer chromatography indicated that allof the starting material had been consumed. The reaction mixture wascooled to ambient temperature. A solid was isolated by filtration,washed with methyl acetate and then dried to provide 28.5 g of2-ethoxymethyl-1-(2-hydroxyethyl)-1H-imidazo[4,5-c]quinoline 5N oxide(crop 1). The filtrate was concentrated under vacuum. The residue wasdiluted with aqueous sodium bicarbonate and then extracted 3 times withdichloromethane. The extracts were combined, washed once with aqueoussodium bicarbonate, washed twice with water, dried with magnesiumsulfate, filtered and then concentrated under vacuum to provide 1.6 g ofadditional product (crop 2).

[0071] Part E

[0072] Crops 1 and 2 from Part D were combined and mixed withdichloromethane (600 mL). Concentrated ammonium hydroxide (450 mL) wasadded. The reaction mixture was cooled in an ice bath and thenp-toluenesulfonyl chloride (22 g) was slowly added to the mixture. Thereaction mixture was stirred at ambient temperature overnight. Thinlayer chromatography indicated the presence of a trace of startingmaterial so 1 g of p-toluenesulfonyl chloride was added and the reactionmixture was stirred for an additional hour. A solid was isolated byfiltration, washed with dichloromethane and then dried to provide 20.2 gof crude 4-amino-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline-1-ethanol. A1 g portion of this material was dissolved in acetone (about 10 mL).Hydrogen chloride/methanol (1 g/5 mL) was added until the solutionbecame acidic. A precipitate formed immediately. The mixture was heatedon a steam bath for 10 minutes. The solid was isolated by filtration,washed with acetone and then recrystallized from methanol/acetone toprovide 0.7 g of4-amino-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline-1-ethanolhydrochloride as an off-white solid. Analysis: Calculated forC₁₅H₁₉CIN₄O: %C, 55.81; %H, 5.93; %N, 17.36; Found: %C, 55.91; %H, 5.90;%N, 17.35.

[0073] Part F

[0074] Thionyl chloride (5 mL) and4-amino-2-ethoxymethyl-1H-imidaz[4,5-c]quinoline-1-ethanol (1 g) werecombined and heated on a steam bath until thin layer chromatography (20%methanol/ethyl acetate) showed the disappearance of starting material.The reaction mixture was cooled to ambient temperature and then slowlypoured into a mixture of ice and water. The mixture was neutralized withsodium bicarbonate and then extracted 3 times with dichloromethane. Theextracts were combined, washed 3 times with aqueous sodium bicarbonate,dried with magnesium sulfate, filtered and then concentrated undervacuum. Acetone (about 10 mL) was added to the residue followed bymethanolic hydrogen chloride (about 1 mL). The mixture was refluxed anda precipitate formed. The reaction mixture was cooled to ambienttemperature. The precipitate-was isolated by filtration and then washedwith acetone. The solid was dissolved in hot methanol and thenprecipitated by the addition of acetone. The precipitate was isolated byfiltration, washed with water and then dried to provide1-(2-chloroethyl)-2-ethoxymethyl-1H-imidazo[4,5-c]quinolin-4-aminehydrochloride. Analysis: Calculated for C₁₅H₁₈CIN₄: %C, 52.80; %H, 5.32;%N, 16.42; Found: %C, 52.67; %H, 5.21; %N, 16.29.

[0075] Part G

[0076] Sodium azide (14.7 g) was added to a solution of1-(2-chloroethyl)-2-ethoxymethyl-1H-imidazo[4,5-c]quinolin-4-amine (22.8g prepared according to the method of Part F) in N,N-dimethylformamide(75 mL). The reaction mixture was heated at reflux for several hours andthen allowed to cool to ambient temperature overnight. The reactionmixture was poured into water (100 ml) and then extracted 3 times withethyl acetate. The extracts were combined, washed 3 times with water,dried over magnesium sulfate, filtered and then concentrated to drynessunder vacuum to provide crude1-(2-azidoethyl)-2-ethoxymethyl-1H-imidazo[4,5-c]quinolin-4-amine as anoil.

[0077] Part H

[0078] Platinum on carbon catalyst was added to a solution of the crudematerial from Part G in ethanol (250 mL). The mixture was reduced on aParr apparatus. The bottle was evacuated several times to removenitrogen and the progress of the reaction was monitored by thin layerchromatography. The reaction mixture was filtered through a layer ofCelite® filter aid to remove the catalyst and the filter pad was washedwith warm ethanol. The filtrate was concentrated under vacuum to providean oil which was purified by column chromatography (silica gel elutingwith methanol/ethyl acetate). An attempt to recrystallize the purifiedoil produced a precipitate which was isolated by filtration. Thefiltrate was concentrated under vacuum to provide an oil. The oil wasdissolved in ethanol. A portion was removed for later use. The remainderwas combined with 10% hydrogen chloride in ethanol and refluxed. Themixture was cooled in an ice bath and then filtered to isolate theresulting solid. The solid was recrystallized from ethanol to provide2-(4-amino-2-ethoxymethyl- 1H-imidazo[4,5-c]quinolin-1yl)ethaneaminetrihydrochloride. Analysis: Calculated for C₁₅H₂₂Cl₃N₅O: %C, 45.64; %H,5.62; %N, 17.74; Found: %C, 46.14; %H, 5.64; %N, 17.83.

Example 1—Preparation of a Labeled Compound of Formula IN-[2-(4-Amino-2-ethoxymethyl-1H-imidazo[4,5-c]quinolin-1-yl)ethyl]-6-[(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino]hexanoamide

[0079] A solution containing6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoicacid, succinimidyl ester (5 mg, BODIPY® FL-X,SE from Molecular Probes)dissolved in dimethyl sulfoxide (1 mL) was combined with2-(4-amino-2-ethoxymethyl-1H-imidazo[4,5-c]quinolin-1-yl)ethanaminetrihydrochloride (2.5 mg). Pyridine (5 drops) was added and the reactionmixture was shaken overnight at ambient temperature. The reactionmixture was purified by high performance liquid chromatography using aBondapak C 18 12.5 nm reverse phase column (available from Waters,Milford, Mass.) eluting with a composite gradient of acetonitrile inwater. In a typical elution, the acetonitrile content was increased from5% to 30% during the initial 15 minutes, followed by a 10 minuteisocratic elution at 30% acetonitrile, a 5 minute gradient to 50%acetonitrile, then a 5 minute isocratic elution at 50% acetonitrile. Allsolvents contained 0.1% trifluoroacetic acid. The fractions containingthe labeled compound were initially identified by comparingchromatograms of the free fluorophore and the unlabeled compound withthat of the reaction mixture. The fractions containing the labeledcompound were then collected, pooled and lyophilized. The labeledcompound had a molecular mass of 672.08 as determined by electrospraymass spectroscopy. The calculated mass is 672.35 based on the proposedempirical formula C₃₅H₄₅N₈O₃BF₂. The uv-visible absorption spectrashowed absorption bands at 505 nm and 325 nm which are characteristic ofthe fluorescent label and of the unlabeled compound respectively.

[0080] The labeled compound of Example 1 and the unlabeled intermediate(2-(4-amino-2-ethoxymethyl-1H-imidazo[4,5-c]quinolin-1-yl)ethaneaminetrihydrochloride) were tested side by side for their ability to inducecytokine biosynthesis using Test Method 1 described above. The compoundof Example 1 was solubilized in ethanol. The unlabeled intermediate wassolubilized in tissue culture water. The results are shown in the tablebelow. Cytokine Induction Labeled Compound Concentration of Example 1Unlabeled Intermediate (μg/ml) TNF (pg/ml) IFN (U/ml) TNF (pg/ml) IFN(U/ml) 10 Not run Not run 2640 140 3 869 62 538 81 1 369 421 49 2878 0.373 2878 0 2878 0.1 46 554 0 421 0.03 42 47 Not run Not run 0.01 0 16 Notrun Not run

Flow Cytometry Analysis

[0081] Whole blood was collected by venipuncture into EDTA vacutainertubes from healthy human donors. PBMCs were separated from whole bloodby ficoll hypaque (Sigma Chemicals, St. Louis, Mo.) density gradientcentrifugation as described in Testerman. The PBMCs were suspended at2×10⁶ cells/mL in RPMI 1640 medium containing 10% fetal bovine serum, 2mM L-glutamine and penicillin/streptomycin solution (RPMI complete). Thecells were then incubated in 12×75 mm polypropylene tubes for 1 hour at37° C. with either the labeled compound of Example 1 or with the BODIPYfluorophore used to prepare the labeled compound. Following incubationthe cells were washed two times with staining buffer (Dulbecco'sPhosphate Buffered Saline without calcium and magnesium, 1% heatinactivated fetal bovine serum, and 0.1% sodium azide). The cells weresuspended in staining buffer and transferred to 12×75 mm polystyrenetubes for analysis by flow cytometry. Binding to mononuclear cells wasdetermined by fluorescence using a FACScan flow cytometer (purchasedfrom Becton Dickinson).

[0082] The histograms of FIGS. 1 and 2 plot the fluorescence intensitywith the more highly fluorescent cell populations being seen further tothe right. The peak area labeled as M3 in the histograms indicates thefluorescence binding to the monocyte population in the peripheral bloodmononuclear cells. These monocytes have been shown to be a major cellproducing cytokines in response to the imidazoquinolines (Gibson et. al.In “Cellular Requirements for Cytokine Production in Response to theImmunomodulators Imiquimod and S-27609”, Journal of Interferon andCytokine Research, 15, 537-545 (1995). The histogram of FIG. 1 wasobtained from cells incubated with the BODIPY fluorophore. The histogramof FIG. 2 was obtained from cells incubated with the BODIPY labeledcompound of Example 1. These histograms demonstrate that the labeledcompound of Example 1 binds to human peripheral blood mononuclear cellswhereas the BODIPY fluorophore by itself does not and that monocytesbind more effectively than other PBMCs

[0083] The labeled compound of Example 1 did not show significantbinding to monocytes when incubated with human PBMCs at 4° C. for 1hour. A histogram similar to that of FIG. 1 was obtained indicating thatbinding is likely intracellular.

Preparation of an Unlabeled Compound of Formula IV4-(4-Amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl)butaneamine

[0084] Part A

[0085] Phosphorous oxychloride (30 mL, 0.32 mole) was slowly added overa period of 1 hour to a solution of 3-nitroquinolin4-ol (50 g, 0.26mole) in N,N-dimethylformamide (150 mL). The reaction mixture was heatedon a steam bath for half an hour and then poured over a mixture of iceand water. The resulting solid was isolated by filtration and thensuspended in chloroform (750 mL). The suspension was heated on a steambath and then filtered while still hot. The filtrate was poured into aseparatory funnel and the chloroform layer was separated from theresidual water. Triethylamine (29 mL) was added to the chloroform layerfollowed by the slow addition of tert-butyl N-(4-aminobutyl)carbamate.The reaction was monitored by thin layer chromatography. When all of thestarting material was gone, the reaction mixture was washed with water,dried over magnesium sulfate and then concentrated under vacuum toprovide 66 g of 1,1-dimethylethylN-{4-[(3-nitroquinolin-4-yl)amino]butyl}carbamate as a yellow solid.

[0086] Part B

[0087] Platinum on carbon (3.6 g of 5%) was added to a solution of1,1-dimethylethyl N-{4-[(3-nitroquinolin-4-yl)amino]butyl}carbamate(36.1 g, 100 mmol) in toluene (1.5 L). The mixture was hydrogenated at50 psi (3.5 Kg/cm²) for 3 hours. The reaction mixture was filteredthrough a layer of Celite® filter aid to remove the catalyst. Thefiltrate was concentrated under vacuum to provide 30.1 g of crude1,1-dimethylethyl N-{4-[(3-aminoquinolin-4-yl)amino]butyl}carbamate as agooey orange syrup.

[0088] Part C

[0089] Under a nitrogen atmosphere, a solution of the material from PartB in dichloromethane (1 L) was cooled to 0° C. Triethylamine (13 mL,93.3 mmol)) was added. Methoxypropionyl chloride (11.5 g, 91.2 mmol) wasadded over a period of 10 minutes. The ice bath was removed. After 1hour the reaction mixture was concentrated to provide a pale orangesolid. This material was combined with ethanol (1 L) and triethylamine(39 mL). The mixture was heated at about 75° C. overnight. The reactionmixture was allowed to cool to ambient temperature and then it wasconcentrated under vacuum to provide an oil. The oil was combined withdiethyl ether (750 mL), stirred for about 15 minutes and then filtered.The filtrate was concentrated under vacuum to provide 34.5 g of crude1,1-dimethylethylN-[4-(2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl)butyl]carbamateas a brown syrup.

[0090] Part D

[0091] Under a nitrogen atmosphere, 3-chloroperbenzoic acid (12.86 gof >77%) was added to a solution of 1,1-dimethylethylN-[4-(2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl)butyl]carbamate(21.3 g, 53.5 mmol) in dichloromethane (200 mL). The reaction mixturewas allowed to stir at ambient temperature overnight. Additional3-chloroperbenzoic acid (200 mg of >77%) was added. After about 2 hoursthe reaction mixture was washed with water, aqueous sodium bicarbonate,water and finally with brine. The organic layer was dried over sodiumsulfate and then concentrated under nitrogen to provide 22 g of crude1-{4-[(1,1-dimethylethylcarbonyl)amino]butyl}-2-(2-methoxyethyl)-1H-irnidazo[4,5-c]quinoline-5N-oxide as a sticky syrup.

[0092] Part E

[0093] Concentrated ammonium hydroxide (˜50 mL) was added to a solutionof the material from Part D in dichloromethane (200 mL). Under anitrogen atmosphere, the reaction mixture was cooled to 0° C. Tosylchloride (10.2 g, 53.5 mmol) was added with rapid stirring over a periodof 10 minutes. The ice bath was removed and the reaction mixture wasallowed to stir at ambient temperature. The layers were separated. Theorganic layer was washed with 1% sodium carbonate (3X), water and brine;dried over sodium sulfate and then concentrated under vacuum to provide20.0 g of crude 1,1-dimethylethylN-[4-(4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl)butyl]carbamateas a mustard yellow solid.

[0094] Part F

[0095] A mixture of 1,1-dimethylethylN-[4-(4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl)butyl]carbamate(18.0 g) and hydrogen chloride/ethanol (40 mL of 2M) was heated to about70° C. After 90 minutes another 40 mL of hydrogen chloride/ethanol wasadded. After about an additional hour, the reaction mixture was allowedto cool while being purged with nitrogen to remove excess hydrogenchloride. The reaction mixture was concentrated to near dryness. Theresidue was triturated with diethyl ether. The resulting solid wasisolated by filtration and then dried under high vacuum to provide 15.8g of the dihydrochloride salt as a light brown solid.

[0096] A portion of the salt (10 g) was dissolved in water. The solutionwas adjusted to pH 11 by the addition of ammonium hydroxide and then itwas extracted several times with chloroform. The extracts were combinedand concentrated under vacuum. The residue was slurried with toluene andthen concentrated to dryness (3X) to provide 6.6 g of4-(4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl)butaneamineas a brown/yellow solid.

Example 2—Preparation of a Labeled Compound of Formula I5-{[({4-[4-Amino-2-(2-methoxethyl)-1H-imidazo[4,5-c]quinolin- 1-yl]butyl}amino) carbonthioyl]amino}-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoicAcid

[0097] A solution of4-(4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl)butaneamine(0.11 g, 0.35 mmol) in warm pyridine (2 mL) was slowly added to asolution of fluorescein-5-isothiocyanate (0.138 g, 0.35 mmol) in warmpyridine (2 mL). The reaction mixture was maintained at ambienttemperature overnight. The reaction mixture was quenched with methanol(15 mL) and then stirred for an hour. The resulting solid was isolatedby filtration, slurried with boiling methanol, and then dried to provide0.12 g of the desired product, as an orange solid, m.p. >245°. Analysisby both thin layer chromatography and high performance liquidchromatography indicated pure product. Analysis: Calculated forC₃₈H₃₄N₆O₆S: %C, 64.94; %H, 4.88; %N, 11.96; Found: %C, 61.33; %H, 5.09;%N, 11.39. High resolution mass spectroscopy: TM=703.2339 Da.,MM=703.2315 Da.

Preparation of an Unlabeled Compound of Formula IV2-(4-Amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)ethaneamine

[0098] Part A

[0099] Triethylamine (66.8 g, 0.66 mmol) was added to a solution oftert-butyl (N-2-aminoethyl)carbamate (55.0 g, 0.34 mmol) in anhydrousdichloromethane (500 mL). 4-Chloro-3-nitroquinoline (68.2 g, 0.33 mmol)was slowly added. The reaction mixture exothermed. The reaction mixturewas allowed to stir overnight. The resulting precipitate was isolated byfiltration and rinsed with water to provide a yellow solid. The filtratewas washed with water, dried over magnesium sulfate and thenconcentrated to provide a yellow solid. The two batches of solid werecombined, slurried with hexane, filtered and then dried to provide 101 gof 1,1-dimethylethyl N-{2-[(3-nitroquinolin-4-yl)amino]ethyl }carbamateas a yellow solid.

[0100] Part B

[0101] Platinum on carbon (1.0 g of 10%) and sodium sulfate (2 g) wereadded to a slurry of 1,1-dimethylethylN-{2-[(3-nitroquinolin-4-yl)amino]ethyl}carbamate (100 g, 0.30 mol) intoluene (500 mL). The reaction vessel was placed on a Parr apparatusunder 50 psi (3.5 Kg/cm²) hydrogen pressure overnight at ambienttemperature. The reaction mixture was filtered through a layer ofCelite® filter aid to remove the catalyst. The filtrate was concentratedunder vacuum to provide 73 g of 1,1-dimethylethylN-{2-[(3-aminoquinolin-4-yl)amino]ethyl}carbamate as a dark gold oil.Thin layer chromatography (silica gel; 10% methanol in dichloromethane)analysis indicated that the material was pure.

[0102] Part C

[0103] Trimethylorthovalerate (5.9 g, 36.4 mmol) was added with stirringto a solution of 1,1-dimethylethylN-{2-[(3-aminoquinolin-4-yl)amino]ethyl}carbamate (10.0 g, 33.1 mmol) inanhydrous toluene (100 mL). The reaction mixture was heated to reflux. A10 mL portion of toluene was removed using a Dean Stark trap and thereaction mixture was maintained for 36 hours. An additional 40 mL oftoluene was removed and then the reaction was allowed to cool to ambienttemperature with continued stirring. The resulting precipitate wasisolated by filtration and dried to provide 6.2 g of 1,1-dimethylethylN-[2-(2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)ethyl]carbamate as a tansolid. Thin layer chromatography (silica gel; 10% methanol indichloromethane) analysis indicated that the material was pure.

[0104] Part D

[0105] 3-Chloroperbenzoic acid (5.15 g of 60%, 17.9 mmol) was slowlyadded with vigorous stirring to a solution of 1,1-dimethylethylN-[2-(2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)ethyl]carbamate (6.0 g,16.3 mmol) in chloroform (60 mL). The reaction mixture was maintained atambient temperature overnight and then it was quenched with aqueoussodium carbonate (250 mL of 1%). The layers were separated. The organiclayer was dried over magnesium sulfate and then concentrated undervacuum to provide ˜6.3 g of1-{2-[(1,1-dimethylethylcarbonyl)amino]ethyl}-2-butyl-1H-imidazo[4,5-c]quinoline-5N-oxide as a tan foam.

[0106] Part E

[0107] A solution of1-{2-[(1,1-dimethylethylcarbonyl)amino]ethyl}-2-butyl-1H-imidazo[4,5-c]quinoline-5N-oxide(39 g, 101 mmol) in chloroform (300 mL) was cooled in an ice bath.Trichloroacetyl isocyanate (21 g, 112 mmol) was added with stirring. Thereaction mixture was maintained at ambient temperature overnight. Thereaction mixture was quenched with concentrated ammonium hydroxide (40mL) and then stirred at ambient temperature for 4 hours. Water was addedto the reaction mixture. The layers were separated. The organic layerwas dried over magnesium sulfate and then concentrated under vacuum toprovide a gold oil. This material was recrystallized from 90%isopropanol to prove 30.2 g of 1,1-dimethylethylN-[2-(4-amino-2-butyl-1H-imidazo[4,5-c ]quinolin-1-yl)ethyl]carbamate.

[0108] Part F

[0109] Trifluoroacetic acid (100 mL) was added with stirring to asolution of 1,1 -dimethylethylN-[2-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)ethyl]carbamate(30.0 g, 78.2 mmol) in acetonitrile (100 mL). The reaction mixture wasmaintained at ambient temperature for 24 hours and then it wasconcentrated under vacuum. The residue was dissolved in a minimal amountof water and the pH of the solution was adjusted to pH 13 using 10%sodium hydroxide. The resulting precipitate was isolated by filtrationand dried under high vacuum to provide 18.1 g of2-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)ethaneamine as anoff-white solid, m.p. 196-199° C.

Example 3—Preparation of a Labeled Compound of Formula I5-{[({2-[4-Amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl]ethyl}amino)carbonthioyl]amino}-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic Acid

[0110] A solution of fluorescein-5-isothiocyanate (778 mg, 2.0 mmol) inpyridine (5 mL) was added to a solution of2-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)ethaneamine (566 mg,2.0 mmol) in pyridine (5 mL). The reaction mixture was heated at refluxfor 30 minutes and then poured into water (50 mL). The resulting orangesolid was isolated by filtration, dried under high vacuum and thenrecrystallized from pyridine to provide 0.76 g of the desired product asan orange solid, m.p. >245°. Analysis by high performance liquidchromatography indicated pure product. Analysis: Calculated forC₃₇H₃₂N₆O₅S: %C, 66.06; %H, 4.79; %N, 12.49; Found: %C, 63.03; %H, 4.89;%N, 12.59. High resolution mass spectroscopy: TM=673.2233 Da.,MM=673.2251 Da.

[0111] The labeled compounds of Examples 2 and 3 were tested for theirability to induce cytokine biosynthesis using Test Method 2 describedabove. The results are shown in the table below where a “+” indicatesthat the compound induced the indicated cytokine at that particularconcentration and a “−” indicates that the compound did not induce theindicated cytokine at that particular concentration. Cytokine InductionConcentration Example 2 Example 3 (μg/ml) TNF IFN TNF IFN 30 + + + +10 + + − + 3.33 + − − − 1.11 − − − − 0.37 − − − − 0.12 − − − − 0.041 − −− − 0.014 − − − −

what is claimed is:
 1. A compound of the formula (I):

wherein: R₁ is a spacer group; R₂ is hydrogen, alkyl, hydroxyalkyl,haloalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, amidoalkyl,alkylamidoalkyl, dialkylamidoalkyl, alkanoylalkyl, azidoalkyl,carbamoylalkyl, alkyl optionally interrupted by a heteroatom; alkenyl,alkenyloxyalkyl; cycloalkylalkyl, heterocycloalkyl; aryl, aralkyl,aralkenyl, heteroarylalkyl, in which aryl is optionally substituted byalkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, halo,amino, alkylamino or dialkylamino; aroylalkyl, or heteroaroylalkyl; R₃and R₄ are each independently hydrogen, alkyl, alkoxy of 1 to 4 carbonatoms, halo, amino, alkylamino, dialkylamino, or when taken together, R₃and R₄ form a fused aryl or heteroaryl group that is optionallysubstituted by one or more substituents selected from alkyl of 1 to 4carbon atoms, alkoxy of 1 to 4 carbon atoms, halo, amino, alkylamino,dialkylamino, hydroxy and alkoxymethyl; or R₃ and R₄ for a fused 5- to7-membered saturated ring, optionally containing one or more heteroatomsand optionally substituted by one or more substituents selected fromalkyl of 1 to 4 carbon atoms, halo or haloalkyl of 1 to 4 carbon atoms;and DYE is a dye moiety, with the proviso that the dye moiety is notdansyl; or a pharmaceutically acceptable acid addition salt thereof. 2.The compound of claim 1 wherein R₁ has the following structure:


3. The compound of claim 1 wherein R₁ has the following structure:


4. The compound of claim 1 wherein DYE is a fluorescent dye moiety. 5.The compound of claim 4 wherein the fluorescent dye moiety is selectedfrom the group consisting of dipyrrometheneboron difluoride dyes,fluorescein, fluorescein derivatives, rhodamine, rhodamine derivativesand Texas Red.
 6. The compound of claim 5 wherein the fluorescent dyemoiety is a dipyrrometheneboron difluoride dye.
 7. The compound of claim6 wherein DYE has the following structure:


8. The compound of claim 4 wherein the fluorescent dye moiety isfluorescein.
 9. The compound of claim 1 wherein R₃ and R₄ together forma fused aryl group, optionally containing one or more heteroatoms andoptionally substituted by one or more substituents selected from alkylof 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, halo, amino,alkylamino, dialkylamino, hydroxy and alkoxymethyl.
 10. The compound ofclaim 1 wherein R₃ and R₄ together form a benzene ring.
 11. The compoundof claim 1 wherein R₃ and R₄ together form a fused 5- to 7-memberedsaturated ring, optionally containing one or more heteroatoms andoptionally substituted by one or more substituents selected from alkylof 1 to 4 carbon atoms, amino, halo and haloalkyl of 1 to 4 carbonatoms.
 12. The compound of claim 1 wherein R₃ and R₄ are eachindependently hydrogen, a straight or branched alkyl of 1 to 8 carbonatoms, alkoxy of 1 to 4 carbon atoms, halo, amino, alkylamino ordialkylamino.
 13. The compound of claim 1 wherein R₂ is hydrogen, alkylcontaining 1 to 8 carbon atoms, or alkoxyalkyl wherein the alkoxy groupcontains 1 to 4 carbon atoms and the alkyl group contains 1 to 4 carbonatoms.
 14. The compound of claim 1 having the following structure:


15. The compound of claim 1 having the following structure


16. The compound of claim 1 having the following structure